SUMOylation of pancreatic glucokinase regulates its cellular stability and activity - PubMed

SUMOylation increases the catalytic activity of recombinant pancreatic hGK in vitro and in MIN6 β-cells. A, first, hGK was SUMOylated in vitro in the presence or absence of SUMO-1 using the indicated recombinant enzymes, for 1 h at 37 °C by standard assay as described under “Experimental Procedures.” Thereafter, the catalytic activity was measured spectrophotometrically, at 50 m

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glucose, on 0.5 μg of non-SUMOylated and SUMOylated pancreatic hGK (column 1 versus column 2) (**, p = 0.009, n = 6). The activity of hGK was also measured in the absence and presence of SUMO-1 alone (columns 3 and 4, respectively), to exclude any contribution to the activity increase seen in column 2, by SUMO-1 interaction (noncovalent) with hGK. B, first, hGK was incubated in the presence (column 1) or absence (column 2) of SUMO protease (Ulp), for 1 h at 30 °C. Thereafter, both samples were SUMOylated as described under A. The specific GK activity of the SUMOylated sample (column 2) was significantly higher (**, p = 0.0002, n = 6) compared with the SENP-1-treated sample (column 1). A slight inhibition of GK activity by SENP-1 alone (column 3 versus 4) was detected and accounted for (*, p = 0.036, n = 6). C, time course for the in vitro SUMOylation of recombinant pancreatic hGK (1 μ

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) as in A, in the presence or absence of SUMO-1, as indicated. The specific activity was measured spectrophotometrically as in A, and after the indicated SUMOylation time, the activity of GK was significantly higher in the SUMOylated versus the non-SUMOylated sample after 40 min (*, p = 0.028, n = 6) and after 60 min (**, p = 0.0038, n = 6). SUMOylation of samples were controlled by SDS-PAGE and immunoblotting using anti-GK. D, recombinant liver and pancreatic hGK and SUMOylation-deficient mutants of the liver (K345R) and pancreatic (K12R,K13R,K15R (K12,13,15R), K346R, K12R,K13R,K15R,K346R (K12,13,15,346R)) isoforms were SUMOylated in vitro, and their specific activity was determined spectrophotometrically, as in A. (**, p = 0.0002, n = 5). E, SUMO-1 overexpression and conjugation increases the activity of exogenous pancreatic hGK in MIN6 β-cells. MIN6 cells were transiently transfected with the indicated plasmids and lysed by mechanical shear forces. The activity of GK was measured as described under “Experimental Procedures” in 100 μg of total proteins from high speed centrifuged cytosolic fractions and shown to be significantly higher (*, p = 0.039, n = 5) in cells transfected with the SENP1 mutant (inactive) compared with cells transfected with the SENP1 plasmid (active). F, inhibiting deSUMOylation increases the activity of endogenous pancreatic mGK in MIN6 β-cells. Cells were lysed by mechanical shear forces in the presence of control medium (light gray columns) or medium containing 0.5 or 1.0 μ

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SENP inhibitors (dark gray columns), and GK activity was measured as described in E. The activity was shown to be significantly higher (*, p = 0.047, n = 3) in MIN6 cells lysed in the presence of (1 μ

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) SENP inhibitors. Samples in E and F were analyzed by SDS-PAGE and immunoblotting using anti-GK and anti-actin. Mouse hexokinase activity in E and F was measured at 0.5 m

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glucose and subtracted from the GK activity measured at 50 m

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glucose. WB, Western blot.